Anti 5mdc mouse antibody

Immunolocalization of 5mdC was performed as previously described (Solís et al., 2012 (link); Testillano et al., 2013 (link)). Historesin semithin sections were mounted on 3-aminopropyltriethoxysilane- coated slides, denatured with 2N HCl for 45 min, washed in PBS and treated with 5% bovine serum albumin (BSA) in PBS for 10 min, incubated with anti-5mdC mouse antibody (Eurogentec) diluted 1/50 in 1% BSA and Alexa-Fluor-488 anti-mouse IgG antibody (Molecular Probes) diluted 1/25. As negative controls, either DNA denaturation step or first antibody was omitted. Sections were counterstained with 1 mg mL^-1 DAPI (4′,6-diamidino-2-phenylindole) for 10 min and analyzed by confocal laser microscopy (TCS-SP5, Leica). Images of maximum projections were obtained with software running in conjunction with the confocal microscope (Leica software LCS version 2.5). Confocal microscopy analysis was performed using the same laser excitation and sample emission capture settings in all immunofluorescence preparations of each species, rapeseed or barley, allowing an accurate comparison between signals of control and AzaC-treated cells.

Methylated DNA was monitored in samples C, T1, T3, T5, and SR (Fig. 1b) by 5-mdC immunolocalization according to the procedure described by Meijón et al. (2010) (link). Briefly, fixed needles were sectioned at 50 µm thickness using a CH 1510-1 cryomicrotome (Leica Microsystems). The samples were permeabilized, blocked with bovine serum albumin and incubated with anti-5-mdC mouse antibody (Eurogentec, Belgium) diluted 1 : 50 in 1% blocking solution. Alexa Fluor 488-labelled anti-mouse polyclonal antibody (Invitrogen) diluted 1 : 25 was used as secondary antibody for detection of 5-mdC. The slides were counterstained with DAPI. Fluorescence was visualized using a TCS-SP2-AOBS confocal microscope (Leica). Maximal projection from a stack of six slides per sample was acquired using the Fiji software (Schindelin et al., 2012 (link)).